Method for production of transfected cells

ABSTRACT

A method is disclosed for the efficient production of a transfected cell which comprises a step of culturing transfected cells in the presence of a cell-adhesive substance, after injection of a foreign gene into target cells, upon production of the transfected cells by transfer of a foreign gene into the target cells through cell perforation. Also provided are transfected cells produced by the method, and a kit for the production of transfected cells that includes a cell-adhesive substance as an essential component, which kit is suitable for use with the method for the efficient production of transfected cells by cell perforation.

FIELD OF THE INVENTION

The present invention relates to a method for production of transfectedcells, more particularly, a method which makes possible to transfer aforeign gene into target cells with high efficiency in the fields suchas cell technology, genetic engineering, developmental engineering andthe like.

BACKGROUND OF THE INVENTION

As a method for transferring a foreign gene into target cells, there areknown a calcium phosphate method, a DEAE-dextran method, a liposomemethod, an electroporation method, a microinjection method, a particlegun method and the like. All of these methods have both advantages anddisadvantages with respect to manipulation procedures, efficiency,damage on cells and the like. Among these methods, a perforation methodsuch as an electroporation method, a microinjection method, a particlegun method and the like enables easy handling of the cells without usingspecial reagents and provides good transfer efficiency. However, damageto cells by perforation cannot be avoided.

The object of the present invention is to provide a method for improvingthe transfer efficiency when a foreign gene is transferred into targetcells by a perforation method to produce transfected cells.

SUMMARY OF THE INVENTION

The first aspect of the present invention relates to a method for theproduction of transfected cells, which is characterized by comprising astep of culturing the cells in the presence of a cell-adhesivesubstance, after injection of a foreign gene into target cells using aperforation method.

The second aspect of the present invention relates to transfected cellscontaining a foreign gene which are produced by the method of thepresent invention.

The third aspect of the present invention relates to a kit, which isused for a method for the production of transfected cells according tothe first aspect of the present invention and is characterized bycontaining a cell-adhesive substance.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the effect of cell-adhesive polypeptidetreatment on gene transfer efficiency in the transfer of pCAT-controlvector into human epidermoid cancer cell A-431.

FIG. 2 is a graph showing the effect of cell-adhesive polypeptidetreatment on gene transfer efficiency in the transfer of pCAT-controlvector into African green monkey kidney cell COS-7-7.

DETAILED DESCRIPTION OF THE INVENTION

The method of the present invention is characterized by culturing thecell in the presence of a substance having the cell-adhesive activityafter a foreign gene is transferred into target cells using aperforation method.

As used herein, the perforation method means a method for injection of agene by perforating a cell wall, including an electroporation method, amicroinjection method, a particle gun method and the like. Theelectroporation method is as described in, for example, Tanpakushitsu,Kakusan, Koso, volume 31, pages 1591-1603 (1986). The microinjectionmethod is as described in, for example, Cell, volume 22, pages 479-488(1980). The particle gun method is as described in, for example,Technique, volume 3, pages 3-16 (1991). These methods include the knownmethods used for transferring a gene into cells.

As cells to be used in these perforation methods, for example, there areanimal cells which are prepared according to a known method"Shin-Seikagaku Jikkenkoza 18, Saibobaiyogijyutsu", 1st edition (1990),edited by Nippon Seikagakugakkai, published by Tokyo Kagakudojin! andcultured animal cells may be used.

As used herein, a cell-adhesive substance refers to a substance havingthe cell-adhesive, that is, the activity to make target cells adhere toa cell, to an extracellular matrix which is a substance filling a spacebetween cells in the tissue, or to a material such as plastic, glass andthe like. In the present invention, any substances having the activitycan be used as long as they give no adverse effects on transfection oftarget cells. Such the activity is to fix cells, for example, to cultureware covered with a cell-adhesive substance while maintaining the cellin its form, or in a spreaded form, that is, in the changed form afterthe cell has been spreaded in one or more directions.

Attachment between the cell-adhesive substance and the target cell canbe assayed using a conventional method. The method includes, forexample, a method described in Nature, 352:438-441 (1991). Briefly, thecell-adhesive substance covers a plastic dish, and a population of cellsto be assayed is put into a medium and allowing it to stand for 30minutes to 2 hours. After this incubation period, non-adhered cells arerecovered, counted and assayed for viability. Cells adhered to thecell-adhesive substance are recovered using trypsin or a celldissociation buffer (for example, Gibco), counted and assayed forviability. Then, a ratio of adhered cells is calculated and comparedwith a standard control such as a plastic dish covered with bovine serumalbumin (BSA). A combination of cell-adhesive substance/cell can bedetermined by substantial adhesion of the target cell with thecell-adhesive substance assayed. In addition, the cell-spreadingactivity can be determined by observing a change in the form of theadhered cells under a microscope before dissociating cells using trypsinor a cell dissociation buffer, in the above procedures.

Examples of the cell-adhesive substance include, for example, acell-adhesive polypeptide or a functional equivalent thereof and acell-adhesive synthetic polymer.

Examples of the polypeptide to be used in the present invention, havingthe cell-adhering activity include a cell-adhesive polypeptide such asinvasin, polylysine which is not derived from an extracellular matrix, apolypeptide showing the cell-spreading activity described in JP-A2-311498, components of an extracellular matrix such as fibronectin,laminin, collagen, vitronectin, osteopontin, thrombospondin, tenascinand the like. The extracellular matrix components can be prepared from anatural or cultured source by a known method International Journal ofCancer, volume 20, pages 1-5 (1977); Journal of Biological Chemistry,volume 254, pages 9933-9937, (1979); "Zoku-Seikagaku Jikkenkoza", volume6, Saibokokkaku no Kozo to Kino (Structure and Function of CellSkeleton) (last volume), (1st edition) (1986) edited by NipponSeikagakugakkai, published by Tokyo Kagakudojin; Cell Structure andFunction, volume 13, pages 281-292 (1988); Journal of BiologicalChemistry, volume 264, pages 18202-18208 (1989); and Journal ofBiological Chemistry, volume 260, pages 12240-12245 (1985)!. Thecell-adhesive polypeptide may be substantially purified extracellularmatrices exhibiting the cell-adhering activity, substantially purifiedextracellular matrix fragments or a mixture thereof. More particularly,proteins and polypeptides having the cell-adhesive or the cell spreadingactivity, or a functional equivalent thereof, may be used.

As these cell-adhesive polypeptides, substantially purified naturalpolypeptides, polypeptides from enzymatical or chemical degradation ofthe natural polypeptides, or the similar polypeptides made by geneticengineering may be used. Further, materials obtained by modifying thesepolypeptides without impairing the function, that is, the cell-adheringactivity or the cell-spreading activity may be used. In the presentinvention, even if the polypeptide has a deletion, substitution,addition and/or insertion of amino acids in the amino acid sequence of apolypeptide from natural origin, as long as the polypeptide has thedesired cell-adhering activity or the cell-spreading activity, it isreferred to as a functional equivalent of a polypeptide having thenatural amino acid sequence. That is, it is known that naturallyoccurring proteins include proteins of which amino acid sequences havemutations such as deletions, insertions, additions, substitutions andthe like of amino acids, which are due to a modification reaction in theliving body after production or during purification, in addition topolymorphism or mutation of genes encoding those naturally occurringproteins. Regardless of these mutations, there are proteins exhibitingthe physiological and biological activity substantially equivalent tothat of proteins having no mutation. Like this, even when there is astructural difference between polypeptides, as long as they share thecommon main functions, they are called polypeptides having thefunctionally equivalent activity.

This is also true where the above mutations are artificially introducedinto the amino acid sequence of proteins. In this case, a greatervariety of mutants may be made. As long as these mutants exhibit thephysiological activity substantially equivalent to that of proteinshaving no mutations, they are interpreted to be a polypeptide having thefunctionally equivalent activity.

For example, in many cases, a methionine residue which is present at aN-terminus of a protein expressed in Escherichia coli is said to beremoved by action of methionine aminopeptidase, thus, generating bothproteins having a methionine residue or those having no methionineresidue depending upon the kind of proteins. However, whether or not aprotein has a methionine residue does not affect the protein activity inmany cases. In addition, it is known that a polypeptide, a certaincysteine residue of which is substituted with a serine residue in theamino acid sequence of human interleukin-2 (IL-2), retains theinterleukin-2 activity Science, volume 224, page 1431 (1984)!.

Further, upon production of proteins by genetic engineering, proteinsare frequently expressed as fused proteins. For example, in order toincrease an amount of an expressed protein of interest, the protein isexpressed by adding a N-terminal peptide chain derived from anotherprotein to the N-terminal of the protein of interest, or adding asuitable peptide chain to the N-terminus or the C-terminus of theprotein of interest to facilitate purification of the protein ofinterest by using a carrier having the affinity to the added peptidechain.

In this respect, the related biotechnological 10 techniques haveadvanced to a state in which deletion, substitution, addition or othermodification of amino acids in a functional region of the polypeptidecan be routinely carried out. Then, the resulting amino acid sequencemay be routinely screened for the desired cell-adhering activity or thecell-spreading activity according to the above method.

Polypeptides having the cell-adhering activity may be an artificialpolypeptide containing, in the molecule, the amino acid sequencenecessary for the cell-adhering activity, for example, the amino acidsequence may be selected from the amino acid sequence represented by SEQID No:1 (RGDS), the amino acid sequence represented by SEQ ID NO:2 (CS1)and the amino acid sequence represented by SEQ ID NO:6 (central sequenceof laminin, YIGSR). These polypeptides can be prepared in a large amountby a genetic engineering method or a chemical synthesis method and maybe used as a purified polypeptide.

Examples of the artificial polypeptide having, in the molecule, theamino acid sequence represented by SEQ ID NO:1 include a polypeptiderepresented by SEQ ID NO:7 described in JP-A 1-180900. The polypeptidecan be prepared using Escherichia coli HB101/pTF1409 (FERM BP-1939)according to a method described in JP-A 1-180900. In additionpolypeptides represented by their respective sequence ID numbers in thesequence list shown in Table 1 below can be prepared according to agenetic engineering method described in each specification.

In addition, a plasmid pCHV90 contained in Escherichia coli HB101/pCHV90in Table 1 can be prepared using Escherichia coli HB101/pHD101 (FERMBP-2264) and Escherichia coli JM109/pTF7021 (FERM BP-1941) according toa method described in JP-A 5-271291.

                  TABLE 1                                                         ______________________________________                                        Laid Open                                                                              SEQ ID   Living Bacterium                                            Publication                                                                            NO       (Escherichia coli)                                                                           Accession No.                                ______________________________________                                        JP-A 1-206998                                                                          8        JM109/pTF7021  FERM BP-1941                                 JP-A 1-261398                                                                          9        HB101/pTF1801  FERM P-9948                                  JP-A 2-97397                                                                           3        JM109/pTF7221  FERM BP-1915                                 JP-A 2-152990                                                                          10       JM109/pTFB800  FERM BP-2126                                 JP-A 2-311498                                                                          11       HB101/pCH101   FERM BP-2799                                 JP-A 3-59000                                                                           12       JM109/pCF406   FERM P-10837                                 JP-A 3-232898                                                                          13       HB101/pCE102   FERM P-11226                                 JP-A 4-54199                                                                           14       JM109/pTF7520 +                                                                              FERM P-11526                                                   VN-IN.TAA                                                            15       JM109/pTF7520 +                                                                              FERM P-11527                                                   Col.sup.×1                                            JP-A 5-271291                                                                          16       HB101/pCHV179  FERM P-12183                                          17       HB101/pCHV90                                                         18       HB101/pCHV89   FERM P-182                                   JP-A 5-97698                                                                           19       JM109/pTF7520ColV                                                                            FERM BP-5277                                 JP-A 5-178897                                                                          20       JM109/pYMH-CF.A                                                                              FERM BP-5278                                 ______________________________________                                    

Alternatively, artificial polypeptides having, in the molecule, theamino acid sequence represented by SEQ ID NO:1 can be PolyRGDSdescribed, described in JP-A 3-173828, can be synthesized and used.

Examples of artificial polypeptides having, in the molecule, the aminoacid sequence represented by SEQ ID NO:2 include a polypeptiderepresented by SEQ ID NO:4, described in JP-A 2-311498, and thepolypeptide can be prepared by genetic engineering using Escherichiacoli HB101/pHD102 (FERM P-10721) according to a method described in JP-A2-311498. In addition, a polypeptide represented by SEQ ID NO:2 may bechemically synthesized according to a method described in JP-A 3-284700.

Further, examples of artificial polypeptides having, in the molecule,the amino acid sequence represented by SEQ ID NO:2 and the amino acidsequence represented by SEQ ID NO:3 include a polypeptide represented bySEQ ID NO:21 described in JP-A 2-311498 and the polypeptide can beprepared by genetic engineering using Escherichia coli HB101/pCH102(FERM BP-2800) according to a method described in JP-A 2-311498. Inaddition, a polypeptide represented by SEQ ID NO:5 described in JP-A3-284700 is a polypeptide containing, in the molecule, the amino acidsequences of SEQ ID NOs: 1 and 2 and the polypeptide can be prepared bygenetic engineering using Escherichia coli HB101/pCS25 (FERM P-11339)according to a method described in JP-A 3-284700.

As described above, examples of the polypeptides used in the presentinvention are cell-adhesive polypeptides containing, in the molecule,the amino acid sequence represented by SEQ ID NO:1 and/or the amino acidsequence represented by SEQ ID NO:2. As the polypeptide, a polypeptideobtained by covalently binding a polypeptide derived from a celladhesion domain of human fibronectin "Fibronectin", pages 47-121 (1989),edited by Mosher, D. F., published by Academic Press! with a CS1polypeptide derived from the same (ibid), a polypeptide derived from aheparin binding domain (ibid) containing a CS1 polypeptide, or apolypeptide derived from a cell adhesion domain can be used, and theycan be made by genetic engineering, respectively. For example,respective necessary regions are taken out from a vector containing aDNA encoding a cell adhesion domain-derived polypeptide, a vectorcontaining a DNA encoding a CS1 polypeptide, and a vector containing aDNA encoding a heparin binding domain-derived peptide containing a CS1polypeptide, respectively, and they can be used alone or in combinationthereof to make a vector expressing a polypeptide containing, in themolecule, the amino acid sequence represented by SEQ ID NO:1 and/or theamino acid sequence represented by SEQ ID NO:2.

When a polypeptide, where a polypeptide containing, in the molecule, theamino acid sequence represented by SEQ ID NO:1 and a polypeptidecontaining, in the molecule, the amino acid sequence represented by SEQID NO:2 are covalently bound, is made, a covalent bonding betweenpolypeptides may be a direct bonding or an indirect bonding, forexample, an indirect bonding via a spacer. A spacer is an insertionsequence for adjusting an intermolecular distance in each region. As thespacer, an arbitrary peptide chain can be used, for example, a sequenceupstream of a CS1 region in the fibronectin molecule. The spacersequence can be easily introduced therein by genetic engineering.

The cell-adhesive synthetic polymers include the knownpoly-N-p-vinylbenzyl-D-lactoneamide (PVLA) polymer.

In the present invention, the target cell include, but is not limitedto, hematopoietic stem cell, peripheral blood stem cell, umbilical bloodcell, ES cell, lymphocyte, cancer cell and the like.

Examples of the foreign gene include, but are not limited to, nucleicacid selected from nucleic acids encoding proteins, nucleic acidsencoding polypeptides, antisense DNAs, antisense RNAs, ribozymes,nucleic acids encoding intracellular antibodies and pseudogenes (decoygenes). In the present invention, the foreign gene may be inserted intoa vector.

Examples of the vector are retrovirus vector, adenovirus vector,vacciniavirus vector, herpesvirus vector and the like.

According to the present invention a transfected cell with a foreigngene can be obtained with high efficiency by culturing a target cell,into which a foreign gene has been transferred by a perforation methodaccording to a conventional method, in the presence of a cell-adhesivesubstance. A cell culture method may be selected from the known methodsdepending upon the cell used. For example, when cell culturing isperformed in the presence of a cell-adhesive polypeptide, 250 to 2000μg/ml of the cell-adhesive polypeptide may be used in a culture mediumto culture it according to a conventional method.

Particularly, culturing is preferably carried out using a culture warecovered with a cell-adhesive substance. The culture ware refers to anyware normally used for cell culture, for example, a culture dish, aculture ware using a microcarrier, and a culture ware using fibroushollow fibers. The culture ware may be covered with the substance bycoating or spraying. For example, the culture may be easily covered withthe polypeptide by dissolving it in a suitable solution such as aphosphate buffered saline (PBS), adding the solution to the culture wareand allowing it to stand for a suitable period of time. An amount of thepolypeptide with which the culture ware is covered may be selected froma range of 50 to 1000 pmol/cm², suitably 150 to 600 pmol/cm².

Transfected cells which have been cultured in the presence of thecell-adhesive substance can be obtained from a culture according to aconventional method. Thus, transfected cells can be produced with highefficiency.

The resulting transfected cells are useful for the production of usefulsubstances by cells using recombinant DNA techniques, development ofdisease models, gene therapy and the like. Thus, transfected cells canbe produced with high efficiency according to the present invention.

In addition, the present invention can be simply carried out by using akit containing a cell-adhesive substance. The cell-adhesive substance tobe contained in the kit may be in a form of solutions or lyophilizedpowders. The kit may contain a buffer for dissolving or diluting thecell-adhesive substance, a cell culture medium, a cell culture ware andthe like. For example, a transfected cell can be simply produced bypreparing a kit combining polypeptides, PBS for diluting thepolypeptide, a cell culture ware and the like which are used for themethod of the present invention. A reagent contained in the kit may bein liquid form or in lyophilized form.

A perforation method in the present invention can be used byappropriately selecting from an electroporation method, a microinjectionmethod, a particle gun method and the like depending upon the purpose.

The present invention is illustrated by the non-limiting Examples below.

EXAMPLE 1

1. Coating of culture dish with cell-adhesive polypeptide.

A polypeptide represented by SEQ ID NO:3 (hereinafter referred to as"C274"), a polypeptide represented by SEQ ID NO:4 (hereinafter referredto as H296) and a polypeptide represented by SEQ ID NO:5 (hereinafterreferred to as "C·CS1") were each dissolved in a phosphate bufferedsaline (PBS) to 1 μM, respectively, which were then sterilized using a0.22 μm filter (Millex-GV, Millipore).

Each 1 ml/well of these solutions was added to a 24-well polystyreneculture dish (manufactured by Corning), respectively, to coat the dishat 4° C. overnight. These dishes were rinsed with a 500μl/well of aDulbecco's modified minimum basal medium not containing bovine fetalserum prior to the addition of a transformed cell described below.

2. Transfection of cells

Two culture dishes (diameter: 100 mm) of human epidermoid cancer cellA-431 which had been cultured in a Dulbecco's modified minimum basalmedium containing 10% bovine fetal serum were rinsed with 10 ml of aDulbecco's modified minimum basal medium not containing bovine fetalserum, respectively, and 3 ml of PBS containing 0.25% bovine trypsin and0.02% EDTA was added thereto to detach cells from the culture dish. Tothese, 7 ml of a Dulbecco's modified minimum basal medium not containingbovine fetal serum was added, followed by centrifugation at 800 rpm for3 minutes to collect cells. The resulting cells were suspended in 10 mlof a Dulbecco's modified minimum basal medium containing bovine fetalserum, followed by centrifugation at 800 rpm for 3 minutes to collectcells. The resulting cells were combined, suspended in 10 ml of PBS, a3/10 aliquot of the suspension was taken and divided into two equalaliquots, which were centrifuged at 800 rpm for 3 minutes to collectcells, respectively. The resulting cells were suspended again in 10 mlof PBS, followed by centrifugation at 800 rpm for 3 minutes to collecttwo batches of cells. One batch of the resulting cells were suspended in1 ml of PBS containing 15 μg of pCAT-control vector (Promega) which hadbeen aseptically prepared, and placed in an electroporation cuvette forGene Pulser (BioRad), which was allowed to stand in ice for 10 minutes.The other batch of the resulting cells were suspended in 1 ml of PBS,and placed in an electroporation cuvette for Gene Pulser (BioRad), whichwas allowed to 15 stand in ice for 10 minutes. Each batch of cells wasallowed to stand in ice for 10 minutes, and voltage was applied theretoat 250 V and 960 μF. After application, the cells were allowed to standin a cuvette in ice for 10 minutes. Thereafter, the cells were recoveredinto 15 ml 20 of a Dulbecco's modified minimum basal medium containing10% bovine fetal serum, 1 ml/well of which were added to a 24-wellpolystyrene culture dish covered with the above polypeptide. These cellswere cultured at 37° C. in the presence of 5% CO₂ gas overnight, themedium was removed by aspiration, and 1 ml/well of a fresh Dulbecco'smodified minimum basal medium containing 10% bovine fetal serum wasadded thereto, followed by culturing at 38° C. in the presence of 5% CO₂gas overnight.

3. Determination of transfection efficiency (efficiency of genetransfer)

The cultured cells were rinsed three times with 1.25 ml of PBS per well,a lysed cell solution was prepared, and detection of expressed CAT wascarried out using CAT-ELISA kit (manufactured by Boehringer Mannheim)according to a method for using the present kit. Since the present kitused a horseradish peroxidase-labelled secondary antibody and ABTS as asubstrate, a ratio of absorbance at 405 nm/490 nm was determined. Avalue obtained by subtracting a blank value from a value for each groupin a case of addition of pCAT-control vector, using a value for thegroup in a case of no addition of pCAT-control vector uponelectroporation as a blank, was adopted as an amount of expressed CAT.

The results thereof are shown in FIG. 1. That is, FIG. 1 is a figureshowing efficiency of gene transfer into a cell in eachpolypeptide-treatment group, where the ordinate shows non-treated groupand each polypeptide-treatment group and the abscissa shows genetransfer efficiency expressed as a ratio of absorbance at 405 nmrelative to that at 490 nm.

As shown in FIG. 1, an amount of expressed CAT in the culture dish inthe C274, H296 or C·CS1-treatment group is higher as compared with thatin a non-treatment group, demonstrating that efficiency of transfer ofpCAT-control vector into a cell is higher.

EXAMPLE 2

1. Coating of culture dish with cell-adhesive polypeptide

A polypeptide represented by SEQ ID NO:3 (hereinafter referred to as"C274"), a polypeptide represented by SEQ ID NO:4 (hereinafter referredto as "H296") and a polypeptide represented by SEQ ID NO:5 (hereinafterreferred to as "C·CS1") were each dissolved in a phosphate bufferedsaline (PBS) to 1 μM, respectively, which were then sterilized using a0.22 μm filter (Millex-GV, Millipore). 1 ml/well of these solutions wereadded to a 24-well polystyrene culture dish (manufactured by Corning) tocoat the dish at 4° C. overnight, respectively. These dishes were rinsedwith 500 μl/well of a Dulbecco's modified minimum basal medium notcontaining bovine fetal serum prior to addition of a transformed celldescribed below.

2. Transfection of cell

Two culture dishes (diameter: 100 mm) of African green monkey kidneycell COS-7 which had been cultured in a Dulbecco's modified minimumbasal medium containing 10% bovine fetal serum were rinsed with 10 ml ofa Dulbecco's modified minimum basal medium not containing bovine fetalserum, respectively, and 3 ml of PBS containing 0.25% bovine trypsin and0.02% EDTA was added thereto to detach cells from the culture dish. Tothese, 7 ml of a Dulbecco's modified minimum basal medium not containingbovine fetal serum was added, respectively, followed by centrifugationat 800 rpm for 3 minutes to collect cells. The resulting cells weresuspended in 10 ml of a Dulbecco's modified minimum basal mediumcontaining bovine fetal serum, followed by centrifugation at 800 rpm for3 minutes to collect cells. The resulting cells were combined, suspendedin 12 ml of PBS, a 5/6 aliquot of the suspension was taken and dividedinto two equal aliquots, which were centrifuged at 800 rpm for 3 minutesto collect cells, respectively. The resulting cells were suspended in 6ml of PBS, followed by centrifugation at 800 rpm for 3 minutes tocollect two batches of cells. One batch of the resulting cells weresuspended in 1 ml of PBS containing 15 μg of pCAT-control vector(Promega) which had been aseptically prepared, and placed in anelectroporation cuvette for Gene Pulser (BioRad), which was allowed tostand in ice for 10 minutes. The other batch of the resulting cells weresuspended in 1 ml of PBS, and placed in an electroporation cuvette forGene Pulser (BioRad), which was allowed to stand in ice for 10 minutes.Each batch of cells was allowed to stand in ice for 10 minutes, andvoltage was applied thereto at 250 V and 960 μF. After application, thecells were allowed to stand in a cuvette in ice for 10 minutes.Thereafter, the cells were recovered into 15 ml of a Dulbeccol'smodified minimum basal medium containing 10% bovine fetal serum, 1ml/well of the cells were added to a 24-well polystyrene culture dishcovered with the above polypeptide. These cells were cultured at 37° C.in the presence of 5% CO₂ gas overnight, the medium was removed byaspiration, and 1 ml/well of a fresh Dulbecco's modified minimum basalmedium containing 10% bovine fetal serum was added, followed byculturing at 37° C. in the presence of 5% CO₂ gas overnight.

3. Determination of transfection efficiency (efficiency of genetransfer)

The cultured cells were rinsed three times with 1.25 ml of PBS per well,a lysed cell solution was prepared, and detection of expressed CAT wascarried out using CAT-ELISA kit (manufactured by Boehringer Mannheim)according to a method for using the present kit. Since the present kitused a horseradish peroxidase-labelled secondary antibody and ABTS as asubstrate, a ratio of absorbance at 405 nm/490 nm was determined. Avalue obtained by subtracting a blank value from a value for each groupin a case of addition of pCAT-control vector, using a value for thegroup in a case of no addition of pCAT-control vector uponelectroporation as a blank, was adopted as an amount of expressed CAT.The results thereof are shown in FIG. 2. That is, FIG. 2 is a figureshowing efficiency of gene transfer into a cell in eachpolypeptide-treatment group, where the ordinate shows non-treated groupand each polypeptide-treatment group and the abscissa shows genetransfer efficiency expressed as a ratio of absorbance at 405 nmrelative to that at 490 nm.

As shown in FIG. 2, an amount of expressed CAT in the culture dish inthe above C274, H296 or C·CS1-treatment group is higher as compared withthat in a non-treatment group, demonstrating that efficiency of transferof pCAT-control vector into a cell is higher.

EXAMPLE 3

Preparation of kit

A kit for production of gene-transferred cells was made from C274, H296,C·CS1, PBS and a culturing dish as shown in Table 2 below. Reagents A, Band C were prepared so that the above polypeptides were adjusted withPBS to the indicated concentrations shown in the Table. Other componentsused are described in Example 1. In addition, all reagents A, B and Cand a diluent for the reagents were aseptically prepared bypre-filtering with a 0.22 μm sterile filter.

                  TABLE 2                                                         ______________________________________                                        Kit for production of transfected cell                                        ______________________________________                                        Reagent A . . . 100 μM C274                                                                        150    μl                                          Reagent B . . . 100 μM H296                                                                        150    μl                                          Reagent C . . . 100 μM C.CS1                                                                       150    μl                                          Diluent for reagents . . . PBS                                                                        45     ml                                             24-well polystyrene culture dish                                                                      3                                                     ______________________________________                                    

As described above, the present invention can overcome the problems ofthe previous methods for gene transfer into cells and provide a method,for production of transfected cells, having improved efficiency of genetransfer into target cells. The present invention can also provide akit, for production of transfected cells, which is used for the method.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 21                                                 (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ArgGlyAspSer                                                                  (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AspGluLeuProGlnLeuValThrLeuProHisProAsnLeuHis                                 151015                                                                        GlyProGluIleLeuAspValProSerThr                                                2025                                                                          (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 274 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ProThrAspLeuArgPheThrAsnIleGlyProAspThrMetArg                                 151015                                                                        ValThrTrpAlaProProProSerIleAspLeuThrAsnPheLeu                                 202530                                                                        ValArgTyrSerProValLysAsnGluGluAspValAlaGluLeu                                 354045                                                                        SerIleSerProSerAspAsnAlaValValLeuThrAsnLeuLeu                                 505560                                                                        ProGlyThrGluTyrValValSerValSerSerValTyrGluGln                                 657075                                                                        HisGluSerThrProLeuArgGlyArgGlnLysThrGlyLeuAsp                                 808590                                                                        SerProThrGlyIleAspPheSerAspIleThrAlaAsnSerPhe                                 95100105                                                                      ThrValHisTrpIleAlaProArgAlaThrIleThrGlyTyrArg                                 110115120                                                                     IleArgHisHisProGluHisPheSerGlyArgProArgGluAsp                                 125130135                                                                     ArgValProHisSerArgAsnSerIleThrLeuThrAsnLeuThr                                 140145150                                                                     ProGlyThrGluTyrValValSerIleValAlaLeuAsnGlyArg                                 155160165                                                                     GluGluSerProLeuLeuIleGlyGlnGlnSerThrValSerAsp                                 170175180                                                                     ValProArgAspLeuGluValValAlaAlaThrProThrSerLeu                                 185190195                                                                     LeuIleSerTrpAspAlaProAlaValThrValArgTyrTyrArg                                 200205210                                                                     IleThrTyrGlyGluThrGlyGlyAsnSerProValGlnGluPhe                                 215220225                                                                     ThrValProGlySerLysSerThrAlaThrIleSerGlyLeuLys                                 230235240                                                                     ProGlyValAspTyrThrIleThrValTyrAlaValThrGlyArg                                 245250255                                                                     GlyAspSerProAlaSerSerLysProIleSerIleAsnTyrArg                                 260265270                                                                     ThrGluIleAsp                                                                  (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 296 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       AlaIleProAlaProThrAspLeuLysPheThrGlnValThrPro                                 51015                                                                         ThrSerLeuSerAlaGlnTrpThrProProAsnValGlnLeuThr                                 202530                                                                        GlyTyrArgValArgValThrProLysGluLysThrGlyProMet                                 354045                                                                        LysGluIleAsnLeuAlaProAspSerSerSerValValValSer                                 505560                                                                        GlyLeuMetValAlaThrLysTyrGluValSerValTyrAlaLeu                                 657075                                                                        LysAspThrLeuThrSerArgProAlaGlnGlyValValThrThr                                 808590                                                                        LeuGluAsnValSerProProArgArgAlaArgValThrAspAla                                 95100105                                                                      ThrGluThrThrIleThrIleSerTrpArgThrLysThrGluThr                                 110115120                                                                     IleThrGlyPheGlnValAspAlaValProAlaAsnGlyGlnThr                                 125130135                                                                     ProIleGlnArgThrIleLysProAspValArgSerTyrThrIle                                 140145150                                                                     ThrGlyLeuGlnProGlyThrAspTyrLysIleTyrLeuTyrThr                                 155160165                                                                     LeuAsnAspAsnAlaArgSerSerProValValIleAspAlaSer                                 170175180                                                                     ThrAlaIleAspAlaProSerAsnLeuArgPheLeuAlaThrThr                                 185190195                                                                     ProAsnSerLeuLeuValSerTrpGlnProProArgAlaArgIle                                 200205210                                                                     ThrGlyTyrIleIleLysTyrGluLysProGlySerProProArg                                 215220225                                                                     GluValValProArgProArgProGlyValThrGluAlaThrIle                                 230235240                                                                     ThrGlyLeuGluProGlyThrGluTyrThrIleTyrValIleAla                                 245250255                                                                     LeuLysAsnAsnGlnLysSerGluProLeuIleGlyArgLysLys                                 260265270                                                                     ThrAspGluLeuProGlnLeuValThrLeuProHisProAsnLeu                                 275280285                                                                     HisGlyProGluIleLeuAspValProSerThr                                             290295                                                                        (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 302 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       ProThrAspLeuArgPheThrAsnIleGlyProAspThrMetArg                                 151015                                                                        ValThrTrpAlaProProProSerIleAspLeuThrAsnPheLeu                                 202530                                                                        ValArgTyrSerProValLysAsnGluGluAspValAlaGluLeu                                 354045                                                                        SerIleSerProSerAspAsnAlaValValLeuThrAsnLeuLeu                                 505560                                                                        ProGlyThrGluTyrValValSerValSerSerValTyrGluGln                                 657075                                                                        HisGluSerThrProLeuArgGlyArgGlnLysThrGlyLeuAsp                                 808590                                                                        SerProThrGlyIleAspPheSerAspIleThrAlaAsnSerPhe                                 95100105                                                                      ThrValHisTrpIleAlaProArgAlaThrIleThrGlyTyrArg                                 110115120                                                                     IleArgHisHisProGluHisPheSerGlyArgProArgGluAsp                                 125130135                                                                     ArgValProHisSerArgAsnSerIleThrLeuThrAsnLeuThr                                 140145150                                                                     ProGlyThrGluTyrValValSerIleValAlaLeuAsnGlyArg                                 155160165                                                                     GluGluSerProLeuLeuIleGlyGlnGlnSerThrValSerAsp                                 170175180                                                                     ValProArgAspLeuGluValValAlaAlaThrProThrSerLeu                                 185190195                                                                     LeuIleSerTrpAspAlaProAlaValThrValArgTyrTyrArg                                 200205210                                                                     IleThrTyrGlyGluThrGlyGlyAsnSerProValGlnGluPhe                                 215220225                                                                     ThrValProGlySerLysSerThrAlaThrIleSerGlyLeuLys                                 230235240                                                                     ProGlyValAspTyrThrIleThrValTyrAlaValThrGlyArg                                 245250255                                                                     GlyAspSerProAlaSerSerLysProIleSerIleAsnTyrArg                                 260265270                                                                     ThrGluIleAspLysProSerAspGluLeuProGlnLeuValThr                                 275280285                                                                     LeuProHisProAsnLeuHisGlyProGluIleLeuAspValPro                                 290295300                                                                     SerThr                                                                        (2) INFORMATION FOR SEQ ID NO: 6:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       TyrIleGlySerArg                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO: 7:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 283 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       AlaValProProProThrAspLeuArgPheThrAsnIleGlyPro                                 151015                                                                        AspThrMetArgValThrTrpAlaProProProSerIleAspLeu                                 202530                                                                        ThrAsnPheLeuValArgTyrSerProValLysAsnGluGluAsp                                 354045                                                                        ValAlaGluLeuSerIleSerProSerAspAsnAlaValValLeu                                 505560                                                                        ThrAsnLeuLeuProGlyThrGluTyrValValSerValSerSer                                 657075                                                                        ValTyrGluGlnHisGluSerThrProLeuArgGlyArgGlnLys                                 808590                                                                        ThrGlyLeuAspSerProThrGlyIleAspPheSerAspIleThr                                 95100105                                                                      AlaAsnSerPheThrValHisTrpIleAlaProArgAlaThrIle                                 110115120                                                                     ThrGlyTyrArgIleArgHisHisProGluHisPheSerGlyArg                                 125130135                                                                     ProArgGluAspArgValProHisSerArgAsnSerIleThrLeu                                 140145150                                                                     ThrAsnLeuThrProGlyThrGluTyrValValSerIleValAla                                 155160165                                                                     LeuAsnGlyArgGluGluSerProLeuLeuIleGlyGlnGlnSer                                 170175180                                                                     ThrValSerAspValProArgAspLeuGluValValAlaAlaThr                                 185190195                                                                     ProThrSerLeuLeuIleSerTrpAspAlaProAlaValThrVal                                 200205210                                                                     ArgTyrTyrArgIleThrTyrGlyGluThrGlyGlyAsnSerPro                                 215220225                                                                     ValGlnGluPheThrValProGlySerLysSerThrAlaThrIle                                 230235240                                                                     SerGlyLeuLysProGlyValAspTyrThrIleThrValTyrAla                                 245250255                                                                     ValThrGlyArgGlyAspSerProAlaSerSerLysProIleSer                                 260265270                                                                     IleAsnTyrArgThrGluIleAspLysProSerGlnMet                                       275280                                                                        (2) INFORMATION FOR SEQ ID NO: 8:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 279 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       ProThrAspLeuArgPheThrAsnIleGlyProAspThrMetArg                                 151015                                                                        ValThrTrpAlaProProProSerIleAspLeuThrAsnPheLeu                                 202530                                                                        ValArgTyrSerProValLysAsnGluGluAspValAlaGluLeu                                 354045                                                                        SerIleSerProSerAspAsnAlaValValLeuThrAsnLeuLeu                                 505560                                                                        ProGlyThrGluTyrValValSerValSerSerValTyrGluGln                                 657075                                                                        HisGluSerThrProLeuArgGlyArgGlnLysThrGlyLeuAsp                                 808590                                                                        SerProThrGlyIleAspPheSerAspIleThrAlaAsnSerPhe                                 95100105                                                                      ThrValHisTrpIleAlaProArgAlaThrIleThrGlyTyrArg                                 110115120                                                                     IleArgHisHisProGluHisPheSerGlyArgProArgGluAsp                                 125130135                                                                     ArgValProHisSerArgAsnSerIleThrLeuThrAsnLeuThr                                 140145150                                                                     ProGlyThrGluTyrValValSerIleValAlaLeuAsnGlyArg                                 155160165                                                                     GluGluSerProLeuLeuIleGlyGlnGlnSerThrValSerAsp                                 170175180                                                                     ValProArgAspLeuGluValValAlaAlaThrProThrSerLeu                                 185190195                                                                     LeuIleSerTrpAspAlaProAlaValThrValArgTyrTyrArg                                 200205210                                                                     IleThrTyrGlyGluThrGlyGlyAsnSerProValGlnGluPhe                                 215220225                                                                     ThrValProGlySerLysSerThrAlaThrIleSerGlyLeuLys                                 230235240                                                                     ProGlyValAspTyrThrIleThrValTyrAlaValThrGlyArg                                 245250255                                                                     GlyAspSerProAlaSerSerLysProIleSerIleAsnTyrArg                                 260265270                                                                     ThrGluIleAspLysProSerGlnMet                                                   275                                                                           (2) INFORMATION FOR SEQ ID NO: 9:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 474 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       AlaValProProProThrAspLeuArgPheThrAsnIleGlyPro                                 151015                                                                        AspThrMetArgValThrTrpAlaProProProSerIleAspLeu                                 202530                                                                        ThrAsnPheLeuValArgTyrSerProValLysAsnGluGluAsp                                 354045                                                                        ValAlaGluLeuSerIleSerProSerAspAsnAlaValValLeu                                 505560                                                                        ThrAsnLeuLeuProGlyThrGluTyrValValSerValSerSer                                 657075                                                                        ValTyrGluGlnHisGluSerThrProLeuArgGlyArgGlnLys                                 808590                                                                        ThrGlyLeuAspSerProThrGlyIleAspPheSerAspIleThr                                 95100105                                                                      AlaAsnSerPheThrValHisTrpIleAlaProArgAlaThrIle                                 110115120                                                                     ThrGlyTyrArgIleArgHisHisProGluHisPheSerGlyArg                                 125130135                                                                     ProArgGluAspArgValProHisSerArgAsnSerIleThrLeu                                 140145150                                                                     ThrAsnLeuThrProGlyThrGluTyrValValSerIleValAla                                 155160165                                                                     LeuAsnGlyArgGluGluSerProLeuLeuIleGlyGlnGlnSer                                 170175180                                                                     ThrValSerAspValProArgAspLeuGluValValAlaAlaThr                                 185190195                                                                     ProThrSerLeuLeuIleSerTrpAspAlaProAlaValThrVal                                 200205210                                                                     ArgTyrTyrArgIleThrTyrGlyGluThrGlyGlyAsnSerPro                                 215220225                                                                     ValGlnGluPheThrValProGlySerLysSerThrAlaThrIle                                 230235240                                                                     SerGlyLeuLysProGlyValAspTyrThrIleThrValTyrAla                                 245250255                                                                     ValThrGlyArgGlyAspSerProAlaSerSerLysProIleSer                                 260265270                                                                     IleAsnTyrArgThrGluIleAspLysProSerGlnAsnGluGly                                 275280285                                                                     LeuAsnGlnProThrAspAspSerCysPheAspProTyrThrVal                                 290295300                                                                     SerHisTyrAlaValGlyAspGluTrpGluArgMetSerGluSer                                 305310315                                                                     GlyPheLysLeuLeuCysGlnCysLeuGlyPheGlySerGlyHis                                 320325330                                                                     PheArgCysAspSerSerArgTrpCysHisAspAsnGlyValAsn                                 335340345                                                                     TyrLysIleGlyGluLysTrpAspArgGlnGlyGluAsnGlyGln                                 350355360                                                                     MetMetSerCysThrCysLeuGlyAsnGlyLysGlyGluPheLys                                 365370375                                                                     CysAspProHisGluAlaThrCysTyrAspAspGlyLysThrTyr                                 380385390                                                                     HisValGlyGluGlnTrpGlnLysGluTyrLeuGlyAlaIleCys                                 395400405                                                                     SerCysThrCysPheGlyGlyGlnArgGlyTrpArgCysAspAsn                                 410415420                                                                     CysArgArgProGlyGlyGluProSerProGluGlyThrThrGly                                 425430435                                                                     GlnSerTyrAsnGlnTyrSerGlnArgTyrHisGlnArgThrAsn                                 440445450                                                                     ThrAsnValAsnCysProIleGluCysPheMetProLeuAspVal                                 455460465                                                                     GlnAlaAspArgGluAspSerArgGlu                                                   470                                                                           (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 385 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      AlaProIleValAsnLysValValThrProLeuSerProProThr                                 151015                                                                        AsnLeuHisLeuGluAlaAsnProAspThrGlyValLeuThrVal                                 202530                                                                        SerTrpGluArgSerThrThrProAspIleThrGlyTyrArgIle                                 354045                                                                        ThrThrThrProThrAsnGlyGlnGlnGlyAsnSerLeuGluGlu                                 505560                                                                        ValValHisAlaAspGlnSerSerCysThrPheAspAsnLeuSer                                 657075                                                                        ProGlyLeuGluTyrAsnValSerValTyrThrValLysAspAsp                                 808590                                                                        LysGluSerValProIleSerAspThrIleIleProAlaValPro                                 95100105                                                                      ProProThrAspLeuArgPheThrAsnIleGlyProAspThrMet                                 110115120                                                                     ArgValThrTrpAlaProProProSerIleAspLeuThrAsnPhe                                 125130135                                                                     LeuValArgTyrSerProValLysAsnGluGluAspValAlaGlu                                 140145150                                                                     LeuSerIleSerProSerAspAsnAlaValValLeuThrAsnLeu                                 155160165                                                                     LeuProGlyThrGluTyrValValSerValSerSerValTyrGlu                                 170175180                                                                     GlnHisGluSerThrProLeuArgGlyArgGlnLysThrGlyLeu                                 185190195                                                                     AspSerProThrGlyIleAspPheSerAspIleThrAlaAsnSer                                 200205210                                                                     PheThrValHisTrpIleAlaProArgAlaThrIleThrGlyTyr                                 215220225                                                                     ArgIleArgHisHisProGluHisPheSerGlyArgProArgGlu                                 230235240                                                                     AspArgValProHisSerArgAsnSerIleThrLeuThrAsnLeu                                 245250255                                                                     ThrProGlyThrGluTyrValValSerIleValAlaLeuAsnGly                                 260265270                                                                     ArgGluGluSerProLeuLeuIleGlyGlnGlnSerThrValSer                                 275280285                                                                     AspValProArgAspLeuGluValValAlaAlaThrProThrSer                                 290295300                                                                     LeuLeuIleSerTrpAspAlaProAlaValThrValArgTyrTyr                                 305310315                                                                     ArgIleThrTyrGlyGluThrGlyGlyAsnSerProValGlnGlu                                 320325330                                                                     PheThrValProGlySerLysSerThrAlaThrIleSerGlyLeu                                 335340345                                                                     LysProGlyValAspTyrThrIleThrValTyrAlaValThrGly                                 350355360                                                                     ArgGlyAspSerProAlaSerSerLysProIleSerIleAsnTyr                                 365370375                                                                     ArgThrGluIleAspLysProSerGlnMet                                                380385                                                                        (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 549 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      ProThrAspLeuArgPheThrAsnIleGlyProAspThrMetArg                                 151015                                                                        ValThrTrpAlaProProProSerIleAspLeuThrAsnPheLeu                                 202530                                                                        ValArgTyrSerProValLysAsnGluGluAspValAlaGluLeu                                 354045                                                                        SerIleSerProSerAspAsnAlaValValLeuThrAsnLeuLeu                                 505560                                                                        ProGlyThrGluTyrValValSerValSerSerValTyrGluGln                                 657075                                                                        HisGluSerThrProLeuArgGlyArgGlnLysThrGlyLeuAsp                                 808590                                                                        SerProThrGlyIleAspPheSerAspIleThrAlaAsnSerPhe                                 95100105                                                                      ThrValHisTrpIleAlaProArgAlaThrIleThrGlyTyrArg                                 110115120                                                                     IleArgHisHisProGluHisPheSerGlyArgProArgGluAsp                                 125130135                                                                     ArgValProHisSerArgAsnSerIleThrLeuThrAsnLeuThr                                 140145150                                                                     ProGlyThrGluTyrValValSerIleValAlaLeuAsnGlyArg                                 155160165                                                                     GluGluSerProLeuLeuIleGlyGlnGlnSerThrValSerAsp                                 170175180                                                                     ValProArgAspLeuGluValValAlaAlaThrProThrSerLeu                                 185190195                                                                     LeuIleSerTrpAspAlaProAlaValThrValArgTyrTyrArg                                 200205210                                                                     IleThrTyrGlyGluThrGlyGlyAsnSerProValGlnGluPhe                                 215220225                                                                     ThrValProGlySerLysSerThrAlaThrIleSerGlyLeuLys                                 230235240                                                                     ProGlyValAspTyrThrIleThrValTyrAlaValThrGlyArg                                 245250255                                                                     GlyAspSerProAlaSerSerLysProIleSerIleAsnTyrArg                                 260265270                                                                     ThrGluIleAspLysProSerMetAlaIleProAlaProThrAsp                                 275280285                                                                     LeuLysPheThrGlnValThrProThrSerLeuSerAlaGlnTrp                                 290295300                                                                     ThrProProAsnValGlnLeuThrGlyTyrArgValArgValThr                                 305310315                                                                     ProLysGluLysThrGlyProMetLysGluIleAsnLeuAlaPro                                 320325330                                                                     AspSerSerSerValValValSerGlyLeuMetValAlaThrLys                                 335340345                                                                     TyrGluValSerValTyrAlaLeuLysAspThrLeuThrSerArg                                 350355360                                                                     ProAlaGlnGlyValValThrThrLeuGluAsnValSerProPro                                 365370375                                                                     ArgArgAlaArgValThrAspAlaThrGluThrThrIleThrIle                                 380385390                                                                     SerTrpArgThrLysThrGluThrIleThrGlyPheGlnValAsp                                 395400405                                                                     AlaValProAlaAsnGlyGlnThrProIleGlnArgThrIleLys                                 410415420                                                                     ProAspValArgSerTyrThrIleThrGlyLeuGlnProGlyThr                                 425430435                                                                     AspTyrLysIleTyrLeuTyrThrLeuAsnAspAsnAlaArgSer                                 440445450                                                                     SerProValValIleAspAlaSerThrAlaIleAspAlaProSer                                 455460465                                                                     AsnLeuArgPheLeuAlaThrThrProAsnSerLeuLeuValSer                                 470475480                                                                     TrpGlnProProArgAlaArgIleThrGlyTyrIleIleLysTyr                                 485490495                                                                     GluLysProGlySerProProArgGluValValProArgProArg                                 500505510                                                                     ProGlyValThrGluAlaThrIleThrGlyLeuGluProGlyThr                                 515520525                                                                     GluTyrThrIleTyrValIleAlaLeuLysAsnAsnGlnLysSer                                 530535540                                                                     GluProLeuIleGlyArgLysLysThr                                                   545                                                                           (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 422 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      ProThrAspLeuArgPheThrAsnIleGlyProAspThrMetArg                                 151015                                                                        ValThrTrpAlaProProProSerIleAspLeuThrAsnPheLeu                                 202530                                                                        ValArgTyrSerProValLysAsnGluGluAspValAlaGluLeu                                 354045                                                                        SerIleSerProSerAspAsnAlaValValLeuThrAsnLeuLeu                                 505560                                                                        ProGlyThrGluTyrValValSerValSerSerValTyrGluGln                                 657075                                                                        HisGluSerThrProLeuArgGlyArgGlnLysThrGlyLeuAsp                                 808590                                                                        SerProThrGlyIleAspPheSerAspIleThrAlaAsnSerPhe                                 95100105                                                                      ThrValHisTrpIleAlaProArgAlaThrIleThrGlyTyrArg                                 110115120                                                                     IleArgHisHisProGluHisPheSerGlyArgProArgGluAsp                                 125130135                                                                     ArgValProHisSerArgAsnSerIleThrLeuThrAsnLeuThr                                 140145150                                                                     ProGlyThrGluTyrValValSerIleValAlaLeuAsnGlyArg                                 155160165                                                                     GluGluSerProLeuLeuIleGlyGlnGlnSerThrValSerAsp                                 170175180                                                                     ValProArgAspLeuGluValValAlaAlaThrProThrSerLeu                                 185190195                                                                     LeuIleSerTrpAspAlaProAlaValThrValArgTyrTyrArg                                 200205210                                                                     IleThrTyrGlyGluThrGlyGlyAsnSerProValGlnGluPhe                                 215220225                                                                     ThrValProGlySerLysSerThrAlaThrIleSerGlyLeuLys                                 230235240                                                                     ProGlyValAspTyrThrIleThrValTyrAlaValThrGlyArg                                 245250255                                                                     GlyAspSerProAlaSerSerLysProIleSerIleAsnTyrArg                                 260265270                                                                     ThrGluIleAspLysProSerMetAlaAsnGluGlyLeuAsnGln                                 275280285                                                                     ProThrAspAspSerCysPheAspProTyrThrValSerHisTyr                                 290295300                                                                     AlaValGlyAspGluTrpGluArgMetSerGluSerGlyPheLys                                 305310315                                                                     LeuLeuCysGlnCysLeuGlyPheGlySerGlyHisPheArgCys                                 320325330                                                                     AspSerSerArgTrpCysHisAspAsnGlyValAsnTyrLysIle                                 335340345                                                                     GlyGluLysTrpAspArgGlnGlyGluAsnGlyGlnMetMetSer                                 350355360                                                                     CysThrCysLeuGlyAsnGlyLysGlyGluPheLysCysAspPro                                 365370375                                                                     HisGluAlaThrCysTyrAspAspGlyLysThrTyrHisValGly                                 380385390                                                                     GluGlnTrpGlnLysGluTyrLeuGlyAlaIleCysSerCysThr                                 395400405                                                                     CysPheGlyGlyGlnArgGlyTrpArgCysAspAsnCysArgArg                                 410415420                                                                     ProGly                                                                        (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 332 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      ProThrAspLeuArgPheThrAsnIleGlyProAspThrMetArg                                 151015                                                                        ValThrTrpAlaProProProSerIleAspLeuThrAsnPheLeu                                 202530                                                                        ValArgTyrSerProValLysAsnGluGluAspValAlaGluLeu                                 354045                                                                        SerIleSerProSerAspAsnAlaValValLeuThrAsnLeuLeu                                 505560                                                                        ProGlyThrGluTyrValValSerValSerSerValTyrGluGln                                 657075                                                                        HisGluSerThrProLeuArgGlyArgGlnLysThrGlyLeuAsp                                 808590                                                                        SerProThrGlyIleAspPheSerAspIleThrAlaAsnSerPhe                                 95100105                                                                      ThrValHisTrpIleAlaProArgAlaThrIleThrGlyTyrArg                                 110115120                                                                     IleArgHisHisProGluHisPheSerGlyArgProArgGluAsp                                 125130135                                                                     ArgValProHisSerArgAsnSerIleThrLeuThrAsnLeuThr                                 140145150                                                                     ProGlyThrGluTyrValValSerIleValAlaLeuAsnGlyArg                                 155160165                                                                     GluGluSerProLeuLeuIleGlyGlnGlnSerThrValSerAsp                                 170175180                                                                     ValProArgAspLeuGluValValAlaAlaThrProThrSerLeu                                 185190195                                                                     LeuIleSerTrpAspAlaProAlaValThrValArgTyrTyrArg                                 200205210                                                                     IleThrTyrGlyGluThrGlyGlyAsnSerProValGlnGluPhe                                 215220225                                                                     ThrValProGlySerLysSerThrAlaThrIleSerGlyLeuLys                                 230235240                                                                     ProGlyValAspTyrThrIleThrValTyrAlaValThrGlyArg                                 245250255                                                                     GlyAspSerProAlaSerSerLysProIleSerIleAsnTyrArg                                 260265270                                                                     ThrGluIleAspLysProSerMetAlaAsnSerAspSerGluCys                                 275280285                                                                     ProLeuSerHisAspGlyTyrCysLeuHisAspGlyValCysMet                                 290295300                                                                     TyrIleGluAlaLeuAspLysTyrAlaCysAsnCysValValGly                                 305310315                                                                     TyrIleGlyGluArgCysGlnTyrArgAspLeuLysTrpTrpGlu                                 320325330                                                                     LeuArg                                                                        (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 341 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      ProThrAspLeuArgPheThrAsnIleGlyProAspThrMetArg                                 151015                                                                        ValThrTrpAlaProProProSerIleAspLeuThrAsnPheLeu                                 202530                                                                        ValArgTyrSerProValLysAsnGluGluAspValAlaGluLeu                                 354045                                                                        SerIleSerProSerAspAsnAlaValValLeuThrAsnLeuLeu                                 505560                                                                        ProGlyThrGluTyrValValSerValSerSerValTyrGluGln                                 657075                                                                        HisGluSerThrProLeuArgGlyArgGlnLysThrGlyLeuAsp                                 808590                                                                        SerProThrGlyIleAspPheSerAspIleThrAlaAsnSerPhe                                 95100105                                                                      ThrValHisTrpIleAlaProArgAlaThrIleThrGlyTyrArg                                 110115120                                                                     IleArgHisHisProGluHisPheSerGlyArgProArgGluAsp                                 125130135                                                                     ArgValProHisSerArgAsnSerIleThrLeuThrAsnLeuThr                                 140145150                                                                     ProGlyThrGluTyrValValSerIleValAlaLeuAsnGlyArg                                 155160165                                                                     GluGluSerProLeuLeuIleGlyGlnGlnSerThrValSerAsp                                 170175180                                                                     ValProArgAspLeuGluValValAlaAlaThrProThrSerLeu                                 185190195                                                                     LeuIleSerTrpAspAlaProAlaValThrValArgTyrTyrArg                                 200205210                                                                     IleThrTyrGlyGluThrGlyGlyAsnSerProValGlnGluPhe                                 215220225                                                                     ThrValProGlySerLysSerThrAlaThrIleSerGlyLeuLys                                 230235240                                                                     ProGlyValAspTyrThrIleThrValTyrAlaValThrGlyArg                                 245250255                                                                     GlyAspSerProAlaSerSerLysProIleSerIleAsnTyrArg                                 260265270                                                                     ThrGluIleAspLysProSerMetGlyIleTyrIleSerGlyMet                                 275280285                                                                     AlaProArgProSerLeuThrLysLysGlnArgPheArgHisArg                                 290295300                                                                     AsnArgLysGlyTyrArgSerGlnArgGlyHisSerArgGlyArg                                 305310315                                                                     AsnGlnAsnSerArgArgProSerArgAlaMetTrpLeuSerLeu                                 320325330                                                                     PheSerSerLysAsnSerSerSerValProAla                                             335340                                                                        (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 446 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      ProThrAspLeuArgPheThrAsnIleGlyProAspThrMetArg                                 151015                                                                        ValThrTrpAlaProProProSerIleAspLeuThrAsnPheLeu                                 202530                                                                        ValArgTyrSerProValLysAsnGluGluAspValAlaGluLeu                                 354045                                                                        SerIleSerProSerAspAsnAlaValValLeuThrAsnLeuLeu                                 505560                                                                        ProGlyThrGluTyrValValSerValSerSerValTyrGluGln                                 657075                                                                        HisGluSerThrProLeuArgGlyArgGlnLysThrGlyLeuAsp                                 808590                                                                        SerProThrGlyIleAspPheSerAspIleThrAlaAsnSerPhe                                 95100105                                                                      ThrValHisTrpIleAlaProArgAlaThrIleThrGlyTyrArg                                 110115120                                                                     IleArgHisHisProGluHisPheSerGlyArgProArgGluAsp                                 125130135                                                                     ArgValProHisSerArgAsnSerIleThrLeuThrAsnLeuThr                                 140145150                                                                     ProGlyThrGluTyrValValSerIleValAlaLeuAsnGlyArg                                 155160165                                                                     GluGluSerProLeuLeuIleGlyGlnGlnSerThrValSerAsp                                 170175180                                                                     ValProArgAspLeuGluValValAlaAlaThrProThrSerLeu                                 185190195                                                                     LeuIleSerTrpAspAlaProAlaValThrValArgTyrTyrArg                                 200205210                                                                     IleThrTyrGlyGluThrGlyGlyAsnSerProValGlnGluPhe                                 215220225                                                                     ThrValProGlySerLysSerThrAlaThrIleSerGlyLeuLys                                 230235240                                                                     ProGlyValAspTyrThrIleThrValTyrAlaValThrGlyArg                                 245250255                                                                     GlyAspSerProAlaSerSerLysProIleSerIleAsnTyrArg                                 260265270                                                                     ThrGluIleAspLysProSerMetValProGlyPheLysGlyAsp                                 275280285                                                                     MetGlyLeuLysGlyAspArgGlyGluValGlyGlnIleGlyPro                                 290295300                                                                     ArgGlyXaaAspGlyProGluGlyProLysGlyArgAlaGlyPro                                 305310315                                                                     ThrGlyAspProGlyProSerGlyGlnAlaGlyGluLysGlyLys                                 320325330                                                                     LeuGlyValProGlyLeuProGlyTyrProGlyArgGlnGlyPro                                 335340345                                                                     LysGlySerThrGlyPheProGlyPheProGlyAlaAsnGlyGlu                                 350355360                                                                     LysGlyAlaArgGlyValAlaGlyLysProGlyProArgGlyGln                                 365370375                                                                     ArgGlyProThrGlyProArgGlySerArgGlyAlaArgGlyPro                                 380385390                                                                     ThrGlyLysProGlyProLysGlyThrSerGlyGlyAspGlyPro                                 395400405                                                                     ProGlyProProGlyGluArgGlyProGlnGlyProGlnGlyPro                                 410415420                                                                     ValGlyPheProGlyProLysGlyProProGlyProProGlyArg                                 425430435                                                                     MetGlyCysProGlyHisProGlyGlnArgGly                                             440445                                                                        (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 457 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      ProThrAspLeuArgPheThrAsnIleGlyProAspThrMetArg                                 151015                                                                        ValThrTrpAlaProProProSerIleAspLeuThrAsnPheLeu                                 202530                                                                        ValArgTyrSerProValLysAsnGluGluAspValAlaGluLeu                                 354045                                                                        SerIleSerProSerAspAsnAlaValValLeuThrAsnLeuLeu                                 505560                                                                        ProGlyThrGluTyrValValSerValSerSerValTyrGluGln                                 657075                                                                        HisGluSerThrProLeuArgGlyArgGlnLysThrGlyLeuAsp                                 808590                                                                        SerProThrGlyIleAspPheSerAspIleThrAlaAsnSerPhe                                 95100105                                                                      ThrValHisTrpIleAlaProArgAlaThrIleThrGlyTyrArg                                 110115120                                                                     IleArgHisHisProGluHisPheSerGlyArgProArgGluAsp                                 125130135                                                                     ArgValProHisSerArgAsnSerIleThrLeuThrAsnLeuThr                                 140145150                                                                     ProGlyThrGluTyrValValSerIleValAlaLeuAsnGlyArg                                 155160165                                                                     GluGluSerProLeuLeuIleGlyGlnGlnSerThrValSerAsp                                 170175180                                                                     ValProArgAspLeuGluValValAlaAlaThrProThrSerLeu                                 185190195                                                                     LeuIleSerTrpAspAlaProAlaValThrValArgTyrTyrArg                                 200205210                                                                     IleThrTyrGlyGluThrGlyGlyAsnSerProValGlnGluPhe                                 215220225                                                                     ThrValProGlySerLysSerThrAlaThrIleSerGlyLeuLys                                 230235240                                                                     ProGlyValAspTyrThrIleThrValTyrAlaValThrGlyArg                                 245250255                                                                     GlyAspSerProAlaSerSerLysProIleSerIleAsnTyrArg                                 260265270                                                                     ThrGluIleAspLysProSerMetAsnValSerProProArgArg                                 275280285                                                                     AlaArgValThrAspAlaThrGluThrThrIleThrIleSerTrp                                 290295300                                                                     ArgThrLysThrGluThrIleThrGlyPheGlnValAspAlaVal                                 305310315                                                                     ProAlaAsnGlyGlnThrProIleGlnArgThrIleLysProAsp                                 320325330                                                                     ValArgSerTyrThrIleThrGlyLeuGlnProGlyThrAspTyr                                 335340345                                                                     LysIleTyrLeuTyrThrLeuAsnAspAsnAlaArgSerSerPro                                 350355360                                                                     ValValIleAspAlaSerThrAlaIleAspAlaProSerAsnLeu                                 365370375                                                                     ArgPheLeuAlaThrThrProAsnSerLeuLeuValSerTrpGln                                 380385390                                                                     ProProArgAlaArgIleThrGlyTyrIleIleLysTyrGluLys                                 395400405                                                                     ProGlySerProProArgGluValValProArgProArgProGly                                 410415420                                                                     ValThrGluAlaThrIleThrGlyLeuGluProGlyThrGluTyr                                 425430435                                                                     ThrIleTyrValIleAlaLeuLysAsnAsnGlnLysSerGluPro                                 440445450                                                                     LeuIleGlyArgLysLysThr                                                         455                                                                           (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 368 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      ProThrAspLeuArgPheThrAsnIleGlyProAspThrMetArg                                 151015                                                                        ValThrTrpAlaProProProSerIleAspLeuThrAsnPheLeu                                 202530                                                                        ValArgTyrSerProValLysAsnGluGluAspValAlaGluLeu                                 354045                                                                        SerIleSerProSerAspAsnAlaValValLeuThrAsnLeuLeu                                 505560                                                                        ProGlyThrGluTyrValValSerValSerSerValTyrGluGln                                 657075                                                                        HisGluSerThrProLeuArgGlyArgGlnLysThrGlyLeuAsp                                 808590                                                                        SerProThrGlyIleAspPheSerAspIleThrAlaAsnSerPhe                                 95100105                                                                      ThrValHisTrpIleAlaProArgAlaThrIleThrGlyTyrArg                                 110115120                                                                     IleArgHisHisProGluHisPheSerGlyArgProArgGluAsp                                 125130135                                                                     ArgValProHisSerArgAsnSerIleThrLeuThrAsnLeuThr                                 140145150                                                                     ProGlyThrGluTyrValValSerIleValAlaLeuAsnGlyArg                                 155160165                                                                     GluGluSerProLeuLeuIleGlyGlnGlnSerThrValSerAsp                                 170175180                                                                     ValProArgAspLeuGluValValAlaAlaThrProThrSerLeu                                 185190195                                                                     LeuIleSerTrpAspAlaProAlaValThrValArgTyrTyrArg                                 200205210                                                                     IleThrTyrGlyGluThrGlyGlyAsnSerProValGlnGluPhe                                 215220225                                                                     ThrValProGlySerLysSerThrAlaThrIleSerGlyLeuLys                                 230235240                                                                     ProGlyValAspTyrThrIleThrValTyrAlaValThrGlyArg                                 245250255                                                                     GlyAspSerProAlaSerSerLysProIleSerIleAsnTyrArg                                 260265270                                                                     ThrGluIleAspLysProSerMetAlaIleAspAlaProSerAsn                                 275280285                                                                     LeuArgPheLeuAlaThrThrProAsnSerLeuLeuValSerTrp                                 290295300                                                                     GlnProProArgAlaArgIleThrGlyTyrIleIleLysTyrGlu                                 305310315                                                                     LysProGlySerProProArgGluValValProArgProArgPro                                 320325330                                                                     GlyValThrGluAlaThrIleThrGlyLeuGluProGlyThrGlu                                 335340345                                                                     TyrThrIleTyrValIleAlaLeuLysAsnAsnGlnLysSerGlu                                 350355360                                                                     ProLeuIleGlyArgLysLysThr                                                      365                                                                           (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 367 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      ProThrAspLeuArgPheThrAsnIleGlyProAspThrMetArg                                 151015                                                                        ValThrTrpAlaProProProSerIleAspLeuThrAsnPheLeu                                 202530                                                                        ValArgTyrSerProValLysAsnGluGluAspValAlaGluLeu                                 354045                                                                        SerIleSerProSerAspAsnAlaValValLeuThrAsnLeuLeu                                 505560                                                                        ProGlyThrGluTyrValValSerValSerSerValTyrGluGln                                 657075                                                                        HisGluSerThrProLeuArgGlyArgGlnLysThrGlyLeuAsp                                 808590                                                                        SerProThrGlyIleAspPheSerAspIleThrAlaAsnSerPhe                                 95100105                                                                      ThrValHisTrpIleAlaProArgAlaThrIleThrGlyTyrArg                                 110115120                                                                     IleArgHisHisProGluHisPheSerGlyArgProArgGluAsp                                 125130135                                                                     ArgValProHisSerArgAsnSerIleThrLeuThrAsnLeuThr                                 140145150                                                                     ProGlyThrGluTyrValValSerIleValAlaLeuAsnGlyArg                                 155160165                                                                     GluGluSerProLeuLeuIleGlyGlnGlnSerThrValSerAsp                                 170175180                                                                     ValProArgAspLeuGluValValAlaAlaThrProThrSerLeu                                 185190195                                                                     LeuIleSerTrpAspAlaProAlaValThrValArgTyrTyrArg                                 200205210                                                                     IleThrTyrGlyGluThrGlyGlyAsnSerProValGlnGluPhe                                 215220225                                                                     ThrValProGlySerLysSerThrAlaThrIleSerGlyLeuLys                                 230235240                                                                     ProGlyValAspTyrThrIleThrValTyrAlaValThrGlyArg                                 245250255                                                                     GlyAspSerProAlaSerSerLysProIleSerIleAsnTyrArg                                 260265270                                                                     ThrGluIleAspLysProSerMetAsnValSerProProArgArg                                 275280285                                                                     AlaArgValThrAspAlaThrGluThrThrIleThrIleSerTrp                                 290295300                                                                     ArgThrLysThrGluThrIleThrGlyPheGlnValAspAlaVal                                 305310315                                                                     ProAlaAsnGlyGlnThrProIleGlnArgThrIleLysProAsp                                 320325330                                                                     ValArgSerTyrThrIleThrGlyLeuGlnProGlyThrAspTyr                                 335340345                                                                     LysIleTyrLeuTyrThrLeuAsnAspAsnAlaArgSerSerPro                                 350355360                                                                     ValValIleAspAlaSerThr                                                         365                                                                           (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 464 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      ProThrAspLeuArgPheThrAsnIleGlyProAspThrMetArg                                 151015                                                                        ValThrTrpAlaProProProSerIleAspLeuThrAsnPheLeu                                 202530                                                                        ValArgTyrSerProValLysAsnGluGluAspValAlaGluLeu                                 354045                                                                        SerIleSerProSerAspAsnAlaValValLeuThrAsnLeuLeu                                 505560                                                                        ProGlyThrGluTyrValValSerValSerSerValTyrGluGln                                 657075                                                                        HisGluSerThrProLeuArgGlyArgGlnLysThrGlyLeuAsp                                 808590                                                                        SerProThrGlyIleAspPheSerAspIleThrAlaAsnSerPhe                                 95100105                                                                      ThrValHisTrpIleAlaProArgAlaThrIleThrGlyTyrArg                                 110115120                                                                     IleArgHisHisProGluHisPheSerGlyArgProArgGluAsp                                 125130135                                                                     ArgValProHisSerArgAsnSerIleThrLeuThrAsnLeuThr                                 140145150                                                                     ProGlyThrGluTyrValValSerIleValAlaLeuAsnGlyArg                                 155160165                                                                     GluGluSerProLeuLeuIleGlyGlnGlnSerThrValSerAsp                                 170175180                                                                     ValProArgAspLeuGluValValAlaAlaThrProThrSerLeu                                 185190195                                                                     LeuIleSerTrpAspAlaProAlaValThrValArgTyrTyrArg                                 200205210                                                                     IleThrTyrGlyGluThrGlyGlyAsnSerProValGlnGluPhe                                 215220225                                                                     ThrValProGlySerLysSerThrAlaThrIleSerGlyLeuLys                                 230235240                                                                     ProGlyValAspTyrThrIleThrValTyrAlaValThrGlyArg                                 245250255                                                                     GlyAspSerProAlaSerSerLysProIleSerIleAsnTyrArg                                 260265270                                                                     ThrGluIleAspLysProSerMetGlyIleArgGlyLeuLysGly                                 275280285                                                                     ThrLysGlyGluLysGlyGluAspGlyPheProGlyPheLysGly                                 290295300                                                                     AspMetGlyIleLysGlyAspArgGlyGluIleGlyProProGly                                 305310315                                                                     ProArgGlyGluAspGlyProGluGlyProLysGlyArgGlyGly                                 320325330                                                                     ProAsnGlyAspProGlyProLeuGlyProProGlyGluLysGly                                 335340345                                                                     LysLeuGlyValProGlyLeuProGlyTyrProGlyArgGlnGly                                 350355360                                                                     ProLysGlySerIleGlyPheProGlyPheProGlyAlaAsnGly                                 365370375                                                                     GluLysGlyGlyArgGlyThrProGlyLysProGlyProArgGly                                 380385390                                                                     GlnArgGlyProThrGlyProArgGlyGluArgGlyProArgGly                                 395400405                                                                     IleThrGlyLysProGlyProLysGlyAsnSerGlyGlyAspGly                                 410415420                                                                     ProAlaGlyProProGlyGluArgGlyProAsnGlyProGlnGly                                 425430435                                                                     ProThrGlyPheProGlyProLysGlyProProGlyProProGly                                 440445450                                                                     LysAspGlyLeuProGlyHisProGlyGlnArgGlyGluThr                                    455460                                                                        (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 432 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      ProThrAspLeuArgPheThrAsnIleGlyProAspThrMetArg                                 151015                                                                        ValThrTrpAlaProProProSerIleAspLeuThrAsnPheLeu                                 202530                                                                        ValArgTyrSerProValLysAsnGluGluAspValAlaGluLeu                                 354045                                                                        SerIleSerProSerAspAsnAlaValValLeuThrAsnLeuLeu                                 505560                                                                        ProGlyThrGluTyrValValSerValSerSerValTyrGluGln                                 657075                                                                        HisGluSerThrProLeuArgGlyArgGlnLysThrGlyLeuAsp                                 808590                                                                        SerProThrGlyIleAspPheSerAspIleThrAlaAsnSerPhe                                 95100105                                                                      ThrValHisTrpIleAlaProArgAlaThrIleThrGlyTyrArg                                 110115120                                                                     IleArgHisHisProGluHisPheSerGlyArgProArgGluAsp                                 125130135                                                                     ArgValProHisSerArgAsnSerIleThrLeuThrAsnLeuThr                                 140145150                                                                     ProGlyThrGluTyrValValSerIleValAlaLeuAsnGlyArg                                 155160165                                                                     GluGluSerProLeuLeuIleGlyGlnGlnSerThrValSerAsp                                 170175180                                                                     ValProArgAspLeuGluValValAlaAlaThrProThrSerLeu                                 185190195                                                                     LeuIleSerTrpAspAlaProAlaValThrValArgTyrTyrArg                                 200205210                                                                     IleThrTyrGlyGluThrGlyGlyAsnSerProValGlnGluPhe                                 215220225                                                                     ThrValProGlySerLysSerThrAlaThrIleSerGlyLeuLys                                 230235240                                                                     ProGlyValAspTyrThrIleThrValTyrAlaValThrGlyArg                                 245250255                                                                     GlyAspSerProAlaSerSerLysProIleSerIleAsnTyrArg                                 260265270                                                                     ThrGluIleAspLysProSerMetAlaAlaGlySerIleThrThr                                 275280285                                                                     LeuProAlaLeuProGluAspGlyGlySerGlyAlaPheProPro                                 290295300                                                                     GlyHisPheLysAspProLysArgLeuTyrCysLysAsnGlyGly                                 305310315                                                                     PhePheLeuArgIleHisProAspGlyArgValAspGlyValArg                                 320325330                                                                     GluLysSerAspProHisIleLysLeuGlnLeuGlnAlaGluGlu                                 335340345                                                                     ArgGlyValValSerIleLysGlyValCysAlaAsnArgTyrLeu                                 350355360                                                                     AlaMetLysGluAspGlyArgLeuLeuAlaSerLysCysValThr                                 365370375                                                                     AspGluCysPhePhePheGluArgLeuGluSerAsnAsnTyrAsn                                 380385390                                                                     ThrTyrArgSerArgLysTyrThrSerTrpTyrValAlaLeuLys                                 395400405                                                                     ArgThrGlyGlnTyrLysLeuGlySerLysThrGlyProGlyGln                                 410415420                                                                     LysAlaIleLeuPheLeuProMetSerAlaLysSer                                          425430                                                                        (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 574 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      ProThrAspLeuArgPheThrAsnIleGlyProAspThrMetArg                                 151015                                                                        ValThrTrpAlaProProProSerIleAspLeuThrAsnPheLeu                                 202530                                                                        ValArgTyrSerProValLysAsnGluGluAspValAlaGluLeu                                 354045                                                                        SerIleSerProSerAspAsnAlaValValLeuThrAsnLeuLeu                                 505560                                                                        ProGlyThrGluTyrValValSerValSerSerValTyrGluGln                                 657075                                                                        HisGluSerThrProLeuArgGlyArgGlnLysThrGlyLeuAsp                                 808590                                                                        SerProThrGlyIleAspPheSerAspIleThrAlaAsnSerPhe                                 95100105                                                                      ThrValHisTrpIleAlaProArgAlaThrIleThrGlyTyrArg                                 110115120                                                                     IleArgHisHisProGluHisPheSerGlyArgProArgGluAsp                                 125130135                                                                     ArgValProHisSerArgAsnSerIleThrLeuThrAsnLeuThr                                 140145150                                                                     ProGlyThrGluTyrValValSerIleValAlaLeuAsnGlyArg                                 155160165                                                                     GluGluSerProLeuLeuIleGlyGlnGlnSerThrValSerAsp                                 170175180                                                                     ValProArgAspLeuGluValValAlaAlaThrProThrSerLeu                                 185190195                                                                     LeuIleSerTrpAspAlaProAlaValThrValArgTyrTyrArg                                 200205210                                                                     IleThrTyrGlyGluThrGlyGlyAsnSerProValGlnGluPhe                                 215220225                                                                     ThrValProGlySerLysSerThrAlaThrIleSerGlyLeuLys                                 230235240                                                                     ProGlyValAspTyrThrIleThrValTyrAlaValThrGlyArg                                 245250255                                                                     GlyAspSerProAlaSerSerLysProIleSerIleAsnTyrArg                                 260265270                                                                     ThrGluIleAspLysProSerMetAlaIleProAlaProThrAsp                                 275280285                                                                     LeuLysPheThrGlnValThrProThrSerLeuSerAlaGlnTrp                                 290295300                                                                     ThrProProAsnValGlnLeuThrGlyTyrArgValArgValThr                                 305310315                                                                     ProLysGluLysThrGlyProMetLysGluIleAsnLeuAlaPro                                 320325330                                                                     AspSerSerSerValValValSerGlyLeuMetValAlaThrLys                                 335340345                                                                     TyrGluValSerValTyrAlaLeuLysAspThrLeuThrSerArg                                 350355360                                                                     ProAlaGlnGlyValValThrThrLeuGluAsnValSerProPro                                 365370375                                                                     ArgArgAlaArgValThrAspAlaThrGluThrThrIleThrIle                                 380385390                                                                     SerTrpArgThrLysThrGluThrIleThrGlyPheGlnValAsp                                 395400405                                                                     AlaValProAlaAsnGlyGlnThrProIleGlnArgThrIleLys                                 410415420                                                                     ProAspValArgSerTyrThrIleThrGlyLeuGlnProGlyThr                                 425430435                                                                     AspTyrLysIleTyrLeuTyrThrLeuAsnAspAsnAlaArgSer                                 440445450                                                                     SerProValValIleAspAlaSerThrAlaIleAspAlaProSer                                 455460465                                                                     AsnLeuArgPheLeuAlaThrThrProAsnSerLeuLeuValSer                                 470475480                                                                     TrpGlnProProArgAlaArgIleThrGlyTyrIleIleLysTyr                                 485490495                                                                     GluLysProGlySerProProArgGluValValProArgProArg                                 500505510                                                                     ProGlyValThrGluAlaThrIleThrGlyLeuGluProGlyThr                                 515520525                                                                     GluTyrThrIleTyrValIleAlaLeuLysAsnAsnGlnLysSer                                 530535540                                                                     GluProLeuIleGlyArgLysLysThrAspGluLeuProGlnLeu                                 545550555                                                                     ValThrLeuProHisProAsnLeuHisGlyProGluIleLeuAsp                                 560565570                                                                     ValProSerThr                                                                  __________________________________________________________________________

We claim:
 1. A method for efficient production of cells transfected bycell perforation comprising the step of culturing target cells in thepresence of a cell-adhesive substance after infection of a foreign geneby cell perforation into the target cells.
 2. The method according toclaim 1, wherein the culturing step is performed in a culture warecovered with the cell-adhesive substance.
 3. The method according toclaim 1, wherein the cell-adhesive substance is a cell-adhesivepolypeptide.
 4. The method according to claim 3, wherein thecell-adhesive polypeptide is a polypeptide having cell-adhering and/orcell-spreading activity.
 5. The method according to claim 3, wherein thepolypeptide having cell-adhering and/or cell-spreading activity is apolypeptide comprising an amino acid sequence represented by SEQ ID NO:1and/or an amino acid sequence represented by SEQ ID NO:2.
 6. The methodaccording to claim 3, wherein the cell-adhesive polypeptide is selectedfrom the group of polypeptides consisting of SEQ ID NOs:3, 4 and
 5. 7.The method according to claim 1, wherein the cell-adhesive substance ispoly-N-p-vinylbenzyl-D-lactoneamide.
 8. The method according to claim 1,wherein the target cells are selected from the group consisting ofhematopoietic stem cells, peripheral blood stem cells, umbilical bloodcells, ES cells, lymphocyte and cancer cells.
 9. The method according toclaim 1, wherein the foreign gene is a nucleic acid selected from thegroup consisting of nucleic acids encoding proteins, nucleic acidsencoding polypeptides, antisense DNAs, antisense RNAs, ribozymes,nucleic acids encoding intracellular antibodies and pseudogenes.
 10. Themethod according to claim 1, wherein the foreign gene is a nucleic acidselected from the group consisting of nucleic acids encoding proteins,nucleic acids encoding polypeptides, antisense DNAs, antisense RNAs,ribozymes, nucleic acids encoding intracellular antibodies andpseudogenes and wherein the nucleic acid is incorporated into a vector.11. The method according to claim 10, wherein the vector is a vectorselected from the group consisting of retrovirus vectors, adenovirusvectors, vacciniavirus vectors and herpesvirus vectors.
 12. The methodaccording to claim 1, wherein cell perforation is performed by a methodselected from the group consisting of an electroporation method, amicroinjection method and a particle gun method.